In a TEM a sample is imaged by passing a beam of energetic electrons with a selectable energy of, for example, between 40 keV and 400 keV, through the sample.
For so-called weak-phase samples, such as biological samples, most electrons pass through the sample while some electrons are elastically or inelastically scattered, the elastically scattered electrons forming diffracted beams. The image is formed by interference of the elastically scattered and unscattered electrons (diffracted and undiffracted beams).
A problem arises in that the Contrast Transfer Function (CTF) for low spatial frequencies in the image is zero or close to zero, resulting in low visibility of large objects/structures.
This is caused by the fact that phase variations induced by the specimen do not lead to intensity variations at the image plane for an in-focus aberration free imaging system. It is noted that, as known to the skilled person, for higher spatial frequencies, lens aberrations can turn phase variations into intensity variations.
A solution to the lack of contrast is the use of a phase plate, the phase plate introducing a phase difference between the diffracted beams and the undiffracted beam. There are two main types of phase plates: the so-called Zernike phase plate leaves the undiffracted beam unchanged, and causes a phase shift of the diffracted beams, while the so-called Boersch phase plate shifts the undiffracted beam while leaving the diffracted beams unchanged.
When the phase shift introduced by the phase plate is approximately π/2 or −π/2 the sine-like behavior of the CTF changes to a cosine-like behavior, and thus a maximum contrast (positive or negative) occurs for low spatial frequencies. For a more thorough description of phase plates and other contrast enhancing devices see Nagayama K et al., ‘Phase Contrast Enhancement with Phase Plates in Biological Electron Microscopy’, Microscopy Today, July 2010, Vol. 18, No. 4 (July 2010), pages 10-13, further referred to as Nagayama [-1-].
A problem of both Boersch phase plates and Zernike phase plates is that they require microscopic holes (typically less than 1 μm) for passing the undiffracted beam unhindered and a centering accuracy which is even an order of magnitude better than the hole size. This resulted in the development of self-centering phase plates, the so-called hole-free phase plates (HFPP).
A hole-free thin-film phase plate, further referred to as a HFPP, is first described by Johnson H M, ‘Chapter 4: In-focus phase contrast electron microscopy’, in ‘Principles and Techniques of Electron Microscopy, Vol. 3: Biological applications’, Ed. M. A. Hayat, ISBN-0-442-25674-4, pages 174-176, further referred to as Johnson [-2-].
The phase plate described in Johnson [-2-] is formed by a contamination spot on a continuous carbon film. This spot is formed using the electron beam in high current mode. The contamination spot modifies the thickness of the film, resulting in a thicker part. This film can then, at lower current, be used as a HFPP, where the undiffracted electrons, passing through the thick spot, experience a larger (negative) phase shift than the diffracted electrons passing through the non-contaminated (or less-contaminated) film. The effect of such a HFPP thus resembles the effect of Zernike phase plate, but with the difference that this phase plate gives a negative phase contrast.
Another HFPP is known from Malac M et al., ‘Convenient contrast enhancement by a hole-free phase plate’, Ultramicroscpy 118 (2012), p. 77-89, further referred to as Malac [-3-], describing a thin film of carbon or gold exposed to an electron beam, resulting in positive charging (due to secondary electron emission).
Yet another type of HFPP is known from European patent application No. EP14165529A1 to Buijsse et al., further referred to as Buijsse. This application describes a so-called Volta phase plate where the electronic structure of the thin film is changed by irradiation with a focused beam of electrons, resulting in the local build-up of a vacuum potential.
Such a HFPP is preferably formed from a featureless thin film, so that—when using the HFPP—the HFPP does not introduce artifacts. A problem arises in that, when forming such a self-centering HFPP, the thin film should be located with high precision in the diffraction plane or a plane conjugated thereto: the position of the thin film should coincide with the diffraction plane or a plane conjugated thereto (the so-called on-plane condition) and should not be spatially removed therefrom (a so-called off-plane condition).
Malac [-3-] is not specific how a featureless thin film is aligned with respect to the diffraction plane. Malac [-3-] suffices by mentioning the need to have a cross-over of size smaller than 1 um at the plane of the HFPP (page 80, right column, last paragraph). Also Buijsse is silent about this aspect.
There is a need for a method to accurately align a thin film with respect to the diffraction plane of the microscope. In particular, there is a need to determine and set the on-plane condition using a thin film formed from an amorphous thin film without contamination or markers. Most particular there is a need to determine on-plane condition of a featureless thin film before forming a HFPP of the thin film.
It is an object of the invention to address these issues. More specifically, it is an object of the invention to provide an automated method of conditioning a thin film to form a HFPP.